The Cytoplasmic Membrane - Mechanisms of action of antibiotics and synthetic anti-infective agents

THE CYTOPLASMIC MEMBRANE  

Composition And Susceptibility Of Membranes To Selective Disruption

The integrity of the cytoplasmic membrane is vital for the normal functioning of all cells. Bacterial membranes do not contain sterols and in this respect differ from membranes of fungi and mammalian cells. Fungal membranes contain predominantly ergosterol as the sterol component whereas mammalian cells contain cholesterol. Gram-negative bacteria contain an additional outer-membrane structure that provides a protective penetration barrier to potentially harmful substances, including many antibiotics. The outer membrane has an unusual asymmetric structure in which phospholipids occupy the inner face and the lipopolysaccharide (LPS) occupies the outer face. The outer membrane is attached to the peptidoglycan by proteins and lipoproteins. The stability of all membranes is maintained by a combination of non-covalent interactions between the constituents involving ionic, hydrophobic and hydrogen bonding. The balance of these interactions can be disturbed by the intrusion of molecules (membrane-active agents) which destroy the integrity of the membrane, thereby causing leakage of cytoplasmic contents or impairment of metabolic functions associated with the membrane. Most membrane-active agents that function in this way, e.g. the alcohols, quaternary ammonium compounds and bisbiguanides  have very poor selectivity. They cannot be used systemically because of their damaging effects upon mammalian cells; instead they are used as skin antiseptics, disinfectants and preservatives. A few agents can be used therapeutically: the polymyxins (colistin), which act principally upon the outer membrane of Gram-negative bacteria, and the antifungal polyenes, which act upon fungal membranes. Other antifungal agents, the imidazoles, triazoles and terbinafine act by blocking the synthesis of ergosterol, the major sterol present in fungal membranes.

Polymyxins

Polymyxin E (colistin) is used in the treatment of serious Gram-negative bacterial infections, particularly those caused by Pseudomonas aeruginosa. It binds tightly to the lipid A component of LPS in the outer membrane of Gram-negative bacteria. The outer leaflet of the membrane structure is distorted, segments of which are released and the permeability barrier is destroyed. The polymyxin molecules can then penetrate to the cytoplasmic membrane where they bind to phospholipids, disrupt membrane integrity, and cause irreversible leakage of cytoplasmic components. Their detergent-like properties are a key feature of this membrane-damaging action, which is similar to that of quaternary ammonium compounds. With increasing resistance to the major groups of antibiotics, some multi-resistant organisms (e.g. Acinetobacter species) remain sensitive only to membraneactive agents such as colistin. However, some Gram-negative bacteria produce LPS that does not bind polymyxins (e.g. Bacteroides species and Burkholderia cenocepacia) while resistance can occur in some normally sensitive organisms such as E. coli and Pseudomonas aeruginosa through modification of their LPS structure (e.g. by addition of aminoarabinose or aminoethanol substituents to the lipid A regions of their LPS).

Daptomycin

This negatively charged bactericidal cyclic lipopetide binds to the surface of the Gram-positive bacterial cell membrane. The binding is dependent on calcium ions. The acyl tail portion of the compound inserts itself into the cytoplasmic membrane and drug molecules aggregate together forming channels. The leakage of potassium ions from the cells results in inhibition of macromolecular synthesis and cell death.

Polyenes

Amphotericin B and nystatin are the most commonly used members of this group of antifungal agents. They derive their action from their strong affinity towards sterols, particularly ergosterol. The hydrophobic polyene region binds to the hydrophobic sterol ring system within fungal membranes. In so doing, the hydroxylated portion of the polyene is pulled into the membrane interior, destabilizing the structure and causing leakage of cytoplasmic constituents. It is possible that polyene molecules associate together in the membrane to form aqueous channels. The pattern of leakage is progressive, with small metal ions such as K⁺ leaking first, followed by larger amino acids and nucleotides. The internal pH of the cells falls as K⁺ ions are released, macromolecules are degraded and the cells are killed. The selective antifungal activity of the polyenes is poor, depending on the higher affinity for ergosterol than cholesterol. Kidney damage is a major problem when polyenes are used systemically to treat severe fungal infections. The problem can be reduced, but not eliminated by administration of amphotericin as a lipid complex or liposome.

Imidazoles And Triazoles

The azole antifungal drugs act by inhibiting the synthesis of the sterol components of the fungal membrane (see also Chapter 4). They are inhibitors of one step in the complex pathway of ergosterol synthesis involving the removal of a methyl group from lanosterol (Figure 12.9).

The 14-α-demethylase enzyme responsible is dependent on cytochrome P-450. The imidazoles and triazoles cause rapid defects in fungal membrane integrity due to reduced levels of ergosterol, with loss of cytoplasmic constituents leading to similar effects to the polyenes. The azoles are not entirely specific for fungal ergosterol synthesis and have some action on mammalian sterol metabolism; for example, they reduce testosterone synthesis.

Terbinafine

This synthetic antifungal agent inhibits the enzyme squalene epoxidase at an early stage in fungal sterol biosynthesis. Acting as a structural analogue of squalene, terbinafine causes the accumulation of this unsaturated hydrocarbon, and a decrease in ergosterol in the fungal cell membrane (Figure 12.9).